Melanocortins are a group of small peptides that bind to a family of five known melanocortin receptors (MC1R through MC5R). Cone, Endocr. Rev. 27(7): 736-749 (2006). They are derived from a common precursor protein, pro-opiomelanocortin (POMC), which is expressed in the neurons of the central and peripheral nervous system, and in the pituitary gland. Voisey et al., Curr. Drug Targets 4(7): 586-597 (2003). The proteolytic cleavage of POMC results in α-, β- and γ-melanocortin and adrenocorticotrophic hormone (ACTH), in addition to several other biologically important peptides. Smith and Funder, Endocr. Rev. 9(1): 159-179 (1988).
Of the five known melanocortin receptors, MC3R and MC4R are thought to be expressed predominantly in the mammalian brain, with MC3R being most highly expressed in the arcuate nucleus of the hypothalamus, and MC4R being expressed in the thalamus, hypothalamus, and hippocampus. Cone, Nat. Neurosci. 8(5): 571-578 (2005). C1R is expressed mainly in the periphery where it is found, for example, on melanoma cells and melanocytes and immune cells. In the neuronal system, MC1R is present only on neurons in the periaqueductal grey matter of the midbrain, where it is believed to have a role in controlling pain. MC2R is predominantly expressed in the adrenal cortex, where it controls steroidogenesis. MC5R is found predominantly in peripheral tissues such as the secretory epithelia of many exocrine glands, where it affects secretory and trophic controls.
Melanocortin peptides were initially thought to have a physiological function primarily directed to the control of skin pigmentation. Hadley and Dorr, Peptides 27(4): 921-930 (2006). However, in the last 25 years, many additional biological activities have been attributed to the melanocortins. Melanocortin peptides that are either agonists (activators) or antagonists (inhibitors) have been shown to control many physiological processes, including pigmentation, feeding, overall metabolic rate/energy homeostasis, endocrine and exocrine gland secretion, inflammation, sodium excretion by the kidney, pain sensation, addictive behavior, and sexual drive. Cone, Nat. Neurosci. 8(5): 571-578 (2005); Cone, Endocr. Rev. 27(7): 736-749 (2006).
Melanocortin analogs have been synthesized for the potential regulation and treatment of many conditions, including weight regulation (e.g., obesity, anorexia, and cachexia), hormonal secretion, and hyposecretion of many exocrine glands (e.g., Sjogren's syndrome), immuno-relevant conditions, and sexual dysfunction. Cone, Nat. Neurosci. 8(5): 571-578 (2005); Cone, Endocr. Rev. 27(7): 736-749 (2006); Bazzani et al., Resuscitation 52(1): 109-115 (2002); and Bertonlini et al., Pharmacol. Res. 59(1): 13-47 (2009). However, in regulating these physiological effects, melanocortin analogs have also been shown to cause hypertension. Gruber et al., Hypertension 6: 468-474 (1984); Klein et al. Life Sciences 36: 769-775 (1985). Experimental studies have shown that administration of melanocortin analogs (peptides) increases arterial pressure and heart rate, and can produce cardiac arrhythmias. Gruber and Callahan, Am. J. Physiol. 257: R681-R694 (1989); and unpublished data.
The physiological regulatory effects of a melanocortin peptide are achieved through the melanocortin pharmacophore: His-Phe-Arg-Trp (SEQ ID NO: 1). This pharmacophore is the minimum set of amino acids necessary for melanocortin-regulated activity. Holder and Haskel-Luevano, Med. Res. Rev., 24(3): 325-356 (2004). In general, all melanocortin peptides share the same active core sequence: His-Phe-Arg-Trp, including melanotropin neuropeptides and adrenocorticotropin. The amino acids surrounding this core sequence in naturally occurring melanocortin peptides are believed to affect the relative affinity for a specific melanocortin receptor.
Various non-naturally occurring melanocortin analogs with enhanced affinity for melanocortin receptors have been synthesized. For example, Klemes et al., Biochem. Biophys. Res. Comm. 137(2): 722-728 (1986), synthesized the melanocortin analogs (Ac-Nle-Asp-His-Phe-Arg-Trp) (SEQ ID NO: 2) and (Ac-Nle-Asp-His-D-Phe-Arg-Trp) (SEQ ID NO: 3). These modified analogs show increased potency for melanotropic activity. Many other melanocortin analogs have been identified. See Balse-Srinivasan et al. J. Med. Chem. 46(17): 3728-3733 (2003).
Further examples of melanocortin analogs that have been synthesized, having increased potency, include: Ac-Nle-cyclo-Asp-His-Phe-Arg-Trp-Lys (SEQ ID NO: 4) and Ac-Nle-cyclo-Asp-His-D-Phe-Arg-Trp-Lys (SEQ ID NO: 5); al-Obeidi et al., J. Med. Chem. 32(12): 2555-2561 (1989); Ac-Nle-cyclo-Asp-His-D-Nal-2′-Arg-Trp-Lys (SEQ ID NO: 6) and Ac-cyclo-Cys-Glu-His-D-Nal-2′-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp (SEQ ID NO: 7); Balse-Srinivasan et al., J. Med. Chem., 46(17); 3728-3733 (2003); Ac-Nle-Glu-His-D-Phe-Arg-D-Trp-Gly (SEQ ID NO: 8); al-Obeidi et al., Peptide Res. 2(1): 140-146 (1989); and His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO: 9); Cone, Neurosci. 8(5): 571-578 (2005); Teixido et al., Brain Res. Bull., 73: 103-107 (2007).
To date, there have been few, if any, attempts at structural modifications to reduce melanocortin peptide side effects or enhance melanocortin in vivo activity. Work on enhancing melanocortin activity has mainly been restricted to in vitro studies. However, sequentially improving a peptide-receptor interaction in an isolated system fails to examine the possibility that one is also improving an interaction with another unrelated receptor system. The basis for such an effect is found in the concept of “overlapping pharmacophores.” Basic science has now shown numerous examples in which a natural or synthetic peptide with a (primary) pharmacophore for one receptor class also contains secondary pharmacophore(s) for a different receptor class that overlaps with the primary class. Agnes et al., Peptides 29(8): 1413-1423 (2008); Lee et al., Biopolymers 90(3): 433-438 (2008). When the secondary pharmacophore produces unwanted side effects, this may lead to the false conclusion that there are unacceptable consequences associated with the primary pharmacophore (i.e., a poor “therapeutic window”). Results herein show that a single structural derivatization can improve the activity of two overlapping pharmacophores: a D-Phe7 substitution in ACTH4-10 improves natriuresis (i.e., sodium excretion; a melanocortin 3 receptor mediated action), and enhances cardiovascular activity (an RFamide receptor dependent effect). Gruber et al., Hypertension 6(4): 468-474 (1984).
The existence of “pharmacophores within pharmacophores” is a statistical consequence of the numerous amino acid residue replacements that can be made within a pharmacophore sequence. “Conservative” amino acid substitutions, replacement of one amino acid residue with another of similar chemical properties, are historically based on the maintenance of secondary and tertiary protein structure and function. Vazquez et al., Arch. Biochem. & Biophys. 305(2): 448-453 (1993). Examples of this include lysine for arginine, or aspartic for glutamic acid: i.e., amino acids with similar side chains. However, there is now evidence for “conservative” amino acid substitutions that traverse traditional amino acid class boundaries but still maintain protein or peptide function.
For example, in addition to the traditional classes of naturally occurring amino acids, acidic, basic, neutral, and non-polar; there are cation, anion, and pi (π) classes. Cation-π interactions are an example of a peptide-receptor binding property that can be produced by the attraction between a variety of cationic side chains of different amino acids residues and the center of an aromatic ring. The ring center has a partial negative charge due to the pi orbitals of the surrounding carbon atoms. Ma and Dougherty, Chem. Rev. 97(5): 1303-1324 (1997). For example; while Arg, Lys, and His are basic amino acids; when considered as part of the cation pair (to a π residue), Gln and Asn can be added to this group. Further, while Tyr may be polar compared to non-polar Phe or Trp, all three of these amino acids can serve as the aromatic partner of a cation-π binding pair. Therefore, a major limitation in recognizing a potential pharmacophore (or pharmacophores) in a given peptide sequence is that many amino acids are members of several different classification groups. Depending on the particular binding property in a ligand-receptor interaction, there may be numerous conservative substitutions available for a particular amino acid residue. Thus, unless one knows the precise types of binding that a residue is participating in, conservative substitutions are uncertain.
Given the numerous conservative substitutions that are possible for many amino acid residues, there are statistical limitations in producing large numbers of truly unique peptide pharmacophores. One approach to estimating the total number of pharmacophores that are potentially possible is to use the mathematical formula first proposed by Gamow to deduce the triplet codon for amino acid residue coding in DNA, i.e., what length of DNA base pairs or “code” raised to the power of the number of different DNA bases, will allow for the coding of at least 20 different amino acids? Gamow et al. Advances in biological and medical physics 4: 23-68 (1956). Given that the average linear peptide pharmacophore (analogous to a DNA codon) is 3-4 residues in length, with 6 known classes of amino acids (analogous to DNA base classifications) there are theoretically about ˜700-4000 potential linear peptide pharmacophores. However, since many amino acids are members of more than one class, the number of unique pharmacophores is much less than theoretical calculations would predict.
The law of probability predicts that “synonymous pharmacophores” will naturally occur. Analogous to synonymous words, these different amino acid sequences can manifest very similar binding characteristics at a specific receptor, and thus produce similar biological activities. These pharmacophores may occur in isolation (a sequence variant of a known peptide pharmacophore), or within the sequence of a larger pharmacophore (i.e., producing an overlapping pharmacophore). These predictions can be verified by constructing conservative substitutions in many different pharmacophores (in particular with cation-π substitutions), and then searching for receptor proteins that bind these sequences.
For example, conservative substitutions in the melanocortin pharmacophore produce 64 peptides with potentially synonymous biological activity, and can bind to the same receptor as the classic melanocortin sequence. Masman, et al. Bioorganic Med. Chem. 14(22): 7604-7614 (2006); Masman, et al. Bioorganic Med. Chem. 16(8): 4347-4358 (2008).
An additional example is found in the RFamide class of peptides; peptides ending in an arginine (R)-phenylalanine (F) C-terminal sequence. This sequence is potentially a cation-π binding motif. Ma and Dougherty, Chem. Rev. 97(5): 1303-1324 (1997). That it is a cation-π binding motif is verified by showing that synonyms of the RF pharmacophore based on conservative cation or π amino acid substitutions have equivalent biological activity. Gaus, et al. Biol. Bull. 184: 322-329 (1993).
Because even small (e.g., di-peptide) pharmacophores can have many synonyms, larger pharmacophores may contain smaller known pharmacophores (or their synonyms) within their sequence. The potential for overlapping pharmacophores to be an unrecognized source of unanticipated side effects in drugs is underscored by the evidence for three unique pharmacophores within the melanocortin tetrapeptide sequence, i.e., melanocortin, RFamide, and δ-opioid. Lee et al., Biopolymers 90(3): 433-438 (2008).
Traditional scientific literature searching (e.g., searching scientific databases such as PubMed) fails to reveal many examples of pharmacophore conservative substitutions. One approach for finding these examples is to use an Internet search engine, and search for the actual substituted pharmacophore sequence. This approach uses the three or single letter amino acid designators to construct the pharmacophore, with the entire structure placed in quotations. Using this approach with conservative sequence variants of the melanocortin pharmacophore; e.g., His-Trp-Arg-Phe or His-Phe-Lys-Trp; revealed a binding protein in fungi that may be the phylogenetic precursor of the melanocortin receptor class. Masman et al., Bioorg. Med. Chem. 14(22): 7604-7614 (2006). Using unique structural features, rather than total sequence homology, has become a recognized way to examine protein homology and phylogeny. Zhu et al., Cell. Mol. Life. Sci. 62(19-20): 2257-2269 (2005). Supporting evidence for a fungal melanocortin receptor precursor hypothesis is that the Lys-Pro-Val sequence, an “address” or enhancer sequence in the α-melanocortin structure, serves a similar function to enhance peptide-binding to the fungal protein. Masman et al., Bioorg. Med. Chem. 16(8): 4347-4358 (2008).
An important aspect of “pharmacophores within pharmacophores” is the potential to specifically regulate their activity: i.e., specifically suppress or enhance one of an overlapping pair or group of pharmacophores. Previous studies have described the how to suppress the cardiovascular side effects of a C-terminal peptide pharmacophore, using a metabolically stable C-terminal extension, without adversely affecting the activity of the larger pharmacophore it overlapped with. WO 2011/026015. However, there are many different structural positions for peptide pharmacophores, e.g., at the C or N-terminus or deeper within the sequence of the peptide. While some pharmacophores give undesirable effects, others have therapeutic qualities. Selective regulation would be an extremely useful property in drug development.
While a reduction in a drug's side effects produces a relative increase in therapeutic activity; i.e., an increase in therapeutic index, stabilization of the C- and/or N-terminus can produce an absolute increase in therapeutic activity. For example, catabolism of ACTH (a 30-amino acid residue peptide) produces peptide fragments similar to ACTH4-10. Saez et al., J. Biol. Chem. 250(5): 1683-1689 (1975); Neidle and Kelly, Arch. Biochem. Biophys. 233(1): 115-126 (1984). These data suggest that the C-terminal sequence of ACTH is very sensitive to enzymatic degradation, because 29 C-terminal residues are lost compared to only 4 N-terminal amino acids. Thus, recognizing where to place a metabolically stable terminal extension on a peptide is crucial in the maximal enhancement of overall molecule stability.